A Fluorescent Cage for Supramolecular Sensing of 3‐Nitrotyrosine in Human Blood Serum

Abstract 3‐Nitrotyrosine (NT) is generated by the action of peroxynitrite and other reactive nitrogen species (RNS), and as a consequence it is accumulated in inflammation‐associated conditions. This is particularly relevant in kidney disease, where NT concentration in blood is considerably high. Therefore, NT is a crucial biomarker of renal damage, although it has been underestimated in clinical diagnosis due to the lack of an appropriate sensing method. Herein we report the first fluorescent supramolecular sensor for such a relevant compound: Fluorescence by rotational restriction of tetraphenylethenes (TPE) in a covalent cage is selectively quenched in human blood serum by 3‐nitrotyrosine (NT) that binds to the cage with high affinity, allowing a limit of detection within the reported physiological concentrations of NT in chronic kidney disease.

S5 brought to 0 °C. Then, TFA (10.0 mL, 130.6 mmol) was added. The mixture was stirred for 5 min. Then Et3SiH (0.79 mL, 4.96 mmol) was added and stirred again for another 5 min. The resulting mixture was concentrated in vacuo. The residue was solved in CH2Cl2 and washed with a saturated aqueous solution of NaHCO3. The organic phase was dried with MgSO4, filtered and the solvent was removed in the rotary evaporator. The crude was purified by flash chromatography (SiO2. First: ethyl acetate/hexane, 30:70. Later: methanol/dichloromethane, 1:99) to give the corresponding product 8 (408.6 mg, 88%). 1 Figure S2. Synthesis of cages Based on a procedure optimized by the group, 4 compound 8 (0.0660 g, 0.09 mmol) was solved in CH2Cl2 (18 mL) under inert atmosphere. Then 6 (0.0551 g, 0.09 mmol) and DBU (0.03 mL, 0.18 mmol) were sequentially added. After 1 min of reaction, 18 mL of a saturated aqueous solution of NH4Cl were added and the aqueous phase was extracted with CH2Cl2. Combined organic phases were dried over anhydrous MgSO4, filtered and concentrated in vacuo. The obtained crude mixture was purified by flash chromatography (SiO2, ethyl acetate/hexane, 50:50) to provide the two isomers of the cage (Cage A, less polar: 6.5 mg, 5 %; Cage B, more polar: 13.0 mg, 11 %).

X-ray structure determination
Cage B crystallizes in the monoclinic crystal system with space group P21/c. As noted in the main text of the paper, the connectivity of the structure was determined unambiguously despite the low quality of the crystals and difficulties with the refinement.
Despite several crystallization attempts, all crystals obtained for Cage B were of very low quality and very weakly diffracting. Figure S3 shows the structure of the best of five datasets collected from three different samples. The diffraction intensity dropped off sharply as a function of the diffraction angle, so that I/sigma(I) was well below 3.0 at around 1.0 Å resolution. The data was therefore truncated to 0.92 Å, resulting in a low-precision structure, as reflected in the A alert in the checkcif shown on page S7. One of the S atoms was disordered and was refined over two positions. An incipient disorder seems to be also present in some further S atoms and ester groups resulting in rather elongated ADPs. RIGU and SADI restraints were used to keep a sensible geometry and the ADPs to a reasonable value. Although the final residual values are quite high (R1 = 0.1348 (I > 2σ(I)) and wR2 = 0.4486 for all reflections), the molecular geometry is shown unambiguously and clearly demonstrates the formation of the cage.
The checkcif file is shown on page S7 and the CIF file can be obtained from the authors on request.
Crystal Data for C76H64O16S4 (M =1361.51 g/mol): monoclinic, space group P21/c (no. 14), a = 14.6562(10) Å, b = 18.5340(11) Å, c = 25.141(2) Å, β = 95.367(7)°, V = 6799.2(8) Å3, Z = 4, T = 293(2) K, μ(CuKα) = 1.860 mm-1, Dcalc = 1.330 g/cm3, 19789 reflections measured (7.064° ≤ 2Θ ≤ 113.982°), 9129 unique (Rint = 0.0588, Rsigma = 0.0771) which were used in all calculations. The final R1 was 0.1348 (I > 2σ(I)) and wR2 was 0.4486 (all data) Figure S3. Structure  It is advisable to attempt to resolve as many as possible of the alerts in all categories. Often the minor alerts point to easily fixed oversights, errors and omissions in your CIF or refinement strategy, so attention to these fine details can be worthwhile. In order to resolve some of the more serious problems it may be necessary to carry out additional measurements or structure refinements. However, the purpose of your study may justify the reported deviations and the more serious of these should normally be commented upon in the discussion or experimental section of a paper or in the "special_details" fields of the CIF. checkCIF was carefully designed to identify outliers and unusual parameters, but every test has its limitations and alerts that are not important in a particular case may appear. Conversely, the absence of alerts does not guarantee there are no aspects of the results needing attention. It is up to the individual to critically assess their own results and, if necessary, seek expert advice.

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Validation response form
Please find below a validation response form (VRF) that can be filled in and pasted into your CIF. S10 Datablock exp_356 -ellipsoid plot S11 S12

Absorption
Solid samples of cage A and cage B were dissolved in 2 mL of THF/H2O (2:8) mixture to obtain a final concentration of 10 μM. The molar absorption coefficient ( ) was obtained by the Lambert-Beer law: Where A is the absorbance, c is the concentrations of cage (mol L -1 ), and b is the thickness of the absorption layer (cm). In our case c was 1 cm. UV/Vis spectra of fluorescein 1 µM in H2O and NaOH 0.1 M was also measured for quantum yield calculations. UV−Vis spectroscopy was performed on a Jasco-560 spectrophotometer.

Quantum Yield
Solutions of cage A and B 10 µM each in 2 mL of THF/H2O 2:8 were prepared. Quantum yields of cage A and B were calculated using fluorescein in NaOH 0,1 M as reference and the equation 2 with λexc = 375 nm, where Q is the quantum yield, I is the integrated fluorescence intensity, A is absorption at excitation wavelength and η is the refractive index of the solvent. The decay of the fluorescence of the cages was measured. A pulsed pump laser at 375 nm was selected for the optical excitation and the detection was tuned at the maximum of the emission S18 band, at around 450 nm. The fluorescence decay curve was not monoexponential. However, the decay curve could be fitted to a bi-exponential function of the type : Where, 1 and 2 are the decay constants and A1 and A2 represent the pre-exponential factors. The fitting was made using IRF reconvolution analysis with F900 software (Edinburgh Instruments) and leaving 1 , 2 , A1 and A2 as fitting parameters.
The best fitting for the decay curves of Cage A and B was obtained with the following parameters: An intensity averaged lifetime can be defined from the fitting parameters 5 as: The average lifetimes of Cage A and B are 2.0 ± 0.2 ns and 3.0 ± 0.3 ns, respectively.

Binding of 3-nitrotyrosine to cage B determined by fluorescence quenching
Quenching was studied by increasing the concentration of the 3-nitrotyrosine (NT) quencher while keeping the concentration of cage B constant at 1 µM. The concentration of NT was increased from 0 µM to 25 µM in steps of 2.5 µM with a total of eleven different solutions with a final volume of 2 mL. The solvent used was 2:8 THF/H2O. Fluorescence emission spectra were obtained in this case using the excitation wavelength at 320 nm.
The plot of F0/F versus NT concentration allows us to obtain an association constant from the slope of the linear fit of 15990 ± 619.58 M -1 in 2:8 THF/H2O.

Limit of detection based on the standard deviation of the blank
In order to obtain the  value based on the blank, the integrated emission intensity of a blank sample (F0) was independently measured 20 times: In order to calculate the standard deviation of the ratio F0/F, and considering the propagation of errors, then the value obtained was  = 0.013. Therefore, the limit of detection of cage B is LoD = 3µM rounding up to micromolar units.

Confirmation of the static quenching mechanism
There are two possible quenching mechanisms to explain the linear Stern-Volmer plots: dynamic quenching and static quenching. Dynamic quenching, also known as collisional quenching, occurs when the quencher diffuses to the fluorescent cage during the lifetime of its excited state. The excited cage returns to its ground state without emission of radiation due to contact with the quencher. That is, quenching occurs without any permanent change in the molecules. On the contrary, static quenching happens when a molecular complex is formed between the fluorescent cage and the quencher, and this new complex is nonfluorescent. In order to distinguish which of the two mechanisms is responsible of the cage fluorescence quenching, lifetime measurements can be performed as a function of the CD biomarker quencher.
In the case of dynamic quenching the lifetime of the excited state of the fluorophore decreases with the quencher concentration according to the following equation: Consequently, S20 Therefore, in the case of dynamic quenching a shortening of the lifetime at the same rate of the emission intensity would be expected as the 3-nitrotyrosine (NT) concentration increases.
On the other hand, if static quenching is occurring, a chemical reaction occurs, in which nonfluorescent cage-NT complexes are formed. In this case, the fluorescence detected comes from cages which have not interacted with the quencher and, consequently, their lifetime remains the same.
The decay of the emission intensity of cage B at a concentration of 10 µM was measured at different concentrations of NT. The lifetime remains practically constant with a value of τ = 2.40 ± 0.01 ns and negligible fluctuations. It can be concluded that quenching occurs only by static quenching.

UV-titration.
A UV-titration was performed to measure the association constant by an alternative method. Concentration of the 3-nitrotyrosine (NT) quencher was increased from 0 to 250 µM in THF/water 2:8, while keeping the concentration of cage B constant at 100 µM. Additionally, we performed a non-linear regression by using the free software available at supramolecular.org, 6 and we obtained an even closer value of Ka =(1.45 ± 0.083)·10 4 M -1 http://app.supramolecular.org/bindfit/view/9d6c8a64-5341-4a32-98a8-12dde8986b1d

Binding of 3-nitrotyrosine to cage B determined by fluorescence quenching in human serum
Three different human blood serums were purchased: One from Sigma-ALDRICH, one from SEQENS and another one from BIOWEST.
The complete description of each serum provided by each company can be found below:

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Donor consent: donations proceed from volunteer donors, who agreed their donation may be used in any way the collection center deems appropriate.
Gratuitousness: donations have not been paid.
Anonymity: each donation is anonymous and is associated with a donation number.
Each unit has also been tested and found negative or non-reactive for: Anti-HIV 1+2 and anti-HCV antibodies, HBs antigen, HIV 1 and HCV RNA Syphilitic serology.
Complete donor traceability on file.

BIOWEST
Collected from the source: When searchers choose their serum, an important factor that should be taken into consideration is the source, which also emphasizes the traceability of the serum. Our system of vertical integration allows us to be certain of the origins and traceability of our human serum. The donors are volunteers. Each manufactured batch is rigorously controlled, from the collection of serum in authorized organisms, and throughout all stages of its treatment and production through to final packaging on the authorized organisms and on our premises. The serum is sourced from multiple blood types and multiple genders. The serum is off-the-clot serum, processed from human blood that has had natural coagulation. The serum is collected or imported and treated in agreement with the European regulations.
Filtration: Final Filter Size: 0.2µm x 2 Sterility: All sera are tested for the absence of aerobic and anaerobic bacteria, fungi and yeast. The sterility test is based on the European Pharmacopeia requirements.
Virus Tested: All of our human serum is tested for: -Hepatitis B antigen (HBs Ag) -Hepatitis C virus and antibodies (HCV) -HIV Type 1 virus and antibodies HIV ½ -Syphilis.
Endotoxin: All sera are tested to determine the levels of endotoxins.
BioWest carries out a chromokinetic quantitative test, according to the method D of the European Pharmacopoeia. The endotoxin reagent is standardized against the US reference endotoxin.
Haemoglobin: The haemoglobin level is measured by spectrophotometer.
Osmolality: Determined by a lowered freezing temperature. The osmometer is calibrated against standard solutions.

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Country of Origin: It is the country in which the serum was taken from the donor. BioWest sera are sourced from France, Germany, Poland or USA.

SIGMA-ALDRICH
Product H6914 is prepared from whole blood that is allowed to clot, centrifuged and the serum removed. There is no additional processing required. Product H6914 will contain more growth factors since it is allowed to spontaneously clot and there will be the release of growth factors from many of the cells (WBC, platelets) present.
All donor units are collected in donor centers located in the United States, which are licensed by the FDA. Solutions were prepared in 96-well black polystyrene plates from Thermo Scientific, 56 of which were used for each measurement. The data of each serum was distinguished by their commercial names: SEQENS, BIOWEST and SIGMA. In these 56 wells the maximum volume was always 100 µL and the 2:6:2 ratio of DMSO/buffer PBS 7,4/serum was always kept constant in each well.
In a first round of measurements, the fluorescence intensity data were taken for a plate with serum from SEQENS (SQ-1), another from SIGMA and another from BIOWEST. To reduce the variability of the data due to equipment repeatability, three measurements were made, one at time 0 min after preparation, another at 10 min and another at 20 min. In a second round of S24 measurements, intensity data were taken for three equal plates with SEQENS serum (SQ-2, SQ-3 and SQ-4). Again, 3 measurements were made for each plate (0, 10 and 20 min).
The assay was carried out in a FLUOStar Omega microplate reader (BMG Labtech) with an excitation filter with wavelength of 380 nm, and an emission filter with wavelength of 470 nm. Measures were made with top optic, 1 cycle and 3 flashes per well with each plate. Multichannel (eight) micropipette was used. The lecture of multiwell were made using Omega Control software and data processing was done using MARS 3.31 software. Fluorescence expected is shown in Figure S17

S31
Analysis were performed using Origin 2018 program. T-student test, for intercept and slope, were calculated at a significance level of 5% (α=0.05). The relationship between the different assays was carried out by calculating the correlation coefficient (Pearson's r). It showed a high coefficient of determination in all cases and the T-values determined that both, the slopes and intercepts, are different from zero. The Stern-Volmer plot of F0/F versus NT concentration allows us to obtain an association constant in 2:6:2 DMSO/Buffer PBS 7,4/serum from the slope of the linear fits.
Regressions were made with the average of all the dates from the sera at same concentration of NT and cage B. Error bars correspond to the standard deviation.

Determination of limit of detection in human serum
Limit of detection (LoD) was calculated based on a method reported in previous literature. 8 It is defined as the minimum analyte concentration that provides a reliable signal for the applied analytical method. It was calculated for the experiments carried out in diluted human serum, particularly when cage B concentration was 16 µM. Changes in F0/F at 16 µM of cage B were plotted versus NT as it can be seen in Figure 7 in the manuscript.
Limit of detection was calculated with the following equation: Where σ is the standard error of the linear regression (0.01204) and b is the slope (1.71·10 -3 ). The detection limit is LoD = 2,3 x 10 -5 mol L -1 .
The LoD was also calculated from the mean and standard deviation of the replicate blank readings, Fblank ± sblank, which were obtained from the first column of the tables on pages S25-S30. Then, following the well-known equation 9 : We obtained the minimum detectable signal (FLOD). The formula is a subtraction because of the quenching of the fluorescence. Then we converted such value into the concentration limit of detection using the appropriate regression line.
Depending on the data used (different sera, different concentrations of cage…) we obtained a LoD varying from 18.75 to 40.8 μM which are in the same range than that obtained by the abovementioned methodology.

Binding of cage B with 1,3,5-trinitrotoluene (TNT) in Ethyl Acetate.
Supramolecular binding of cage B with a nitroaromatic guest in a pure organic solvent was also studied, in order to evaluate the relevance of the hydrophobic effect in the association. We chose TNT as guest. Even when it is not 3-nitrotyrosine (NT), it is relatively similar and it could serve as a model to study the binding in organic solvent. As it can be seen in Figures

Selectivity measurements
Different common bioanalytes were tested with cage B to check if they were able to quench the fluorescence of the sensor. In all cases a concentration of 25 μM was used. No quenching was observed for any of the bioanalytes.